Attention is given to the importance of performing analysis or measurement at or near locations where analysis or measurement is required (hereinafter referred to generally as “POC analyses etc.”), such as analysis for bedside diagnosis performing measurement necessary for medical diagnosis near a patient (POC (point of care) analyses), analysis of hazardous substances in rivers and wastes at locations such as rivers, dumping grounds and the like, and inspection of contamination in each location of cooking, harvesting, and importing foods, and emphasis is currently put on the development of detection methods and equipment applied to these POC analyses etc. These POC analyses etc. are required to be performed simply, in a short time and inexpensively.
As for conventional micro analyses methods, GCMS equipment and LCMS equipment for quantifying samples by a mass spectrometer after separating the sample by capillary gas chromatography (CGC), capillary liquid chromatography (CLC) and the like have been generally used. However, these analyzers are not suitable for use in such measurement locations as the bedside of patients, contaminated rivers and dumping grounds because mass spectrometers are large in size and operations are complicated. Furthermore, for analyzers for use in medical diagnosis using blood and the like as samples, it is desirable that sample contacting portions are disposable.
To solve these problems, a concept of analysis method generally called μTAS (micro total analysis system) intended to make conventionally used analyzers smaller and carry out reaction and separation of samples using chips of a few centimeter square including capillaries to perform electrophoresis for simply performing micro analysis has been proposed (Sensors and Actuators, B1 (1990), 244–248, A. Manz et al.). This μTAS has advantages that the amount of samples and reagents required for detection of components and the amount of wastes and effluent waste of consumable items used for detection and the like are reduced, and detection can be done in a short time.
The μTAS is constituted by samples consisting of liquids, gases and the like in the chip (hereinafter referred to as “fluid”), means for transporting reagents and means for achieving their reactions, etc. in addition to the aforesaid chips and analysis methods, for each of which research is being performed. However, each of them has disadvantages as describe below, and a comprehensive μTAS combining all of these components is not completed yet in the present circumstances.
For example, materials forming capillary are generally glass and silicon that can be finely processed with high accuracy (For example, Japanese Patent Laid-Open No. 2-245655), but they also have disadvantages that the process cost is high and careful handling is required because they are apt to break, and the like. Furthermore, as described above, for use in medical diagnosis and the like, it is desirable that chips are disposable because they contact samples originated from patients such as blood etc., but materials such as glass and silicon are incombustible, thus raising problems also in waste treatment. As research intended to solve these problems arising when glass and silicon are used, a method in which chips are produced from resin (R. M. McCormick et al./Anal. Chem. Vol. 69, No. 14 (1997) 2626–2630, Japanese Patent Laid-Open No. 2-259557, Japanese Patent No. 2639087 (Registration: Apr. 25, 1997, Shimadzu Corp.). The method of producing resin chips includes a method in which the surface of Si-wafer is processed applying semiconductor fine processing technology, followed by electrocasting Ni and removing Si by dissolution and the like to fabricate a master processed with resin, and then acrylic resin or the like is injection-molded using the above described master as a matrix to mold chips (Analytical Chemistry 69, 2626–2630 (1997) (Aclara Biosciences)).
In this way, chips made of resin are excellent in disposability and mass-producibility, but have problems as described below if fluorescent methods, absorptiometric methods and the like used in conventional detection equipment are adopted as means for detecting substances in the chip as in case of glass and silicon.
Prior arts will be further described below with emphasis on detection equipment.
Methods of analyzing samples flowing in the capillary generally include fluorescence spectroscopic methods (for example, S. C. Jacobson et al., Anal. Chem. Vol. 66, 4127–4132, 1994, Japanese Patent Laid-Open No. 2-245655), absorptiometric methods (for example, N. Kuroda et al., J. Chrom atogr., Vol. 798, 325–334, 1998), and chemical luminescent methods (for example, M. F. Regehr et al., J. Capillary Electrophor, Vol. 3, 117–124, 1996).
Of these methods, the chemical luminescent method and fluorescent method are methods in which a substance to be detected is changed into a compound in excited state in the presence of a catalyst such as an oxidizer and energy emitted as light when the compound changes from this state to a ground state is detected (in case of fluorescent method, energy is transferred to an energy acceptor coexisting with the excited compound and the energy emitted when this acceptor changes from an excited state to a ground state is detected). On the other hand, the absorptiometric method is a method in which light is introduced in a solution containing a substance to be detected for measuring the intensity of transmitted light and determining the ratio of the intensity of the transmitted light to the intensity of the incoming light. As for sensitivity, it is generally said that the ranking is, from lowest to highest, the absorptiometric method, the fluorescent method and the chemical luminescent method.
As major chemical luminescent reactions, methods by luminol and lucigenine have been known for a long time. Also the chemical luminescent reaction has advantages such as high speed and high sensitivity and relatively inexpensive equipment because no light source is required for detection, but it has disadvantages that luminescence is rapidly decayed, reagents for use are unstable, the background is high and so forth.
In a similar way, the fluorescent method has an advantage that its reaction system has been known for a long time, but it requires the source of excitation light as an optical system and optical filters for separating excitation light and fluorescence, and the like.
Also, these methods using luminescent phenomena have a problem of poor light-intercepting efficiency because emitted light is diverged in all directions. In case of fluorescent method, general versatility is not high because the yield of emitting fluorescence is low and it is necessary to establish a reaction system for converting an object substance to be measured into a limited fluorescent substance.
In particular, in the field of clinical investigation for medical diagnosis, since integration of measured values into those with standard methods defined by academic societies and the like is in progress, substantial changes in measurement systems may raise problems.
Also, the absorptiometric method has a disadvantage that it is necessary to make the length of optical path large so as to obtain accurate results and particularly long optical path is obtained for detecting a trace amount of samples, thereby making the structure of detection cells complicated, because the ratio of incoming light to transmitted light is detected in principle.
In this way, the detection with conventional absorptiometric methods and fluorescent methods using cuvettes and the like can be carried out using relatively small equipment, but the measurement with chips equipped with capillaries intended for application to the POC analyses etc. allows only small length of optical path because the diameter of the capillary is reduced, and only low sensitivity can be obtained.
Methods in which light is not applied to the capillary vertically but is applied in the flow direction in order to make the length of optical path larger have been proposed (for example, Japanese Patent Laid-Open No. 8-304339), but these methods have a disadvantage that detection in the flow direction is not easy in case of capillaries formed on the plane chips and the chip structure and the structure of detecting portions are more complicated.
As another method of detecting a trace amount of components, the photothermal detection method (thermal lens detection method) has been long known in which samples in liquid are excited with an excitation light to form so-called thermal lens and changes in the thermal lens are measured with a detection light (Japanese Patent Laid-Open No. 60-174933, A. C. Boccara et al., Appl. Phys. Lett. 36, 130, 1980).
In the photothermal detection method, a thermal lens with thickness of about 0.1 μm to 1 mm is usually formed by excitation light. In case sufficient length of optical path, for example, about 1 cm can be provided, the photothermal detection method is not usually used because two kinds of light sources, i.e. excitation light and detection light are usually required, in contrast to the absorptiometric method and fluorescent method. Also, the excitation light and detection light are made coaxial and are let in the capillary, thus causing the equipment to be complicated. However, methods in which two lasers are not made coaxial but made to cross or face each other (J. Liquid Chromatography 12, 2575–2585 (1989), Japanese Patent Laid-Open No. 10-142177 (Molecular Biophotonics)) and methods in which one laser is diverged for use and the shift in focal position itself due to photothermal conversion is detected (Japanese Patent Laid-Open No. 4-369467 (Yokogawa Electric Corp.)) have also been proposed.
One example of photothermal detection methods using Ar laser and He—Ne laser is a method in which a sample is placed on a glass plate and sandwiched with another glass plate (Anal. Chem. 65, 2938–2940 (1993)).
Furthermore, there is an example that application has been made from the outside of a plane chip comprising capillaries to an analyzer that sends liquid using pumps. (Analysis No. 4, 280–284, 1997, M. Harada et al., Anal. Chem. Vol. 65, 2938–2940, 1993, Kawanishi et al., Japan Analytical Chemistry, Abstracts of 44th Annual Meeting, p. 119, 1995, etc.)
These photothermal detection methods are mainly intended to improve local absolute sensitivity as of “How many molecules can be detected.” Thus, methods are dominating in which laser is focused as much as possible, the excitation light is concentrated in a small volume and the thermal lens occurring in the micro space is detected.
Furthermore, among these examples, those showing a concept that chemical reaction systems such as reaction tanks, fluid control elements and detecting portions are integrated in a chip (Journal of Japan Mechanics Association 100, 615–617 (1997), Sensor/Actuator/Week 1997 General Symposium Abstracts “Microsensor” Session 3, pp. 19–23 (Apr. 17, 1997)) are also found. Furthermore, in these examples, capillaries are formed and thus glass is used as a material for making grooves on the surface.
In case silicon and glass are used as materials for chips, etching protection coats (Cr etc.) are formed in thickness of several thousand Å on a substrate made of glass, quartz or Si substrate using a technique such as vacuum evaporation, and patterning resists are applied thereon using a spinner. Then, the resist is exposed to ultraviolet light using a mask for photolithography, followed by carrying out development (removing a non-cured portion with a solvent) patterning resulting in a desired shape. Next, using the patterned resist as an etching mask, the etching protection coat is dissolved and removed with potassium ferricyanide solution and the like resulting in patterning. Next, using the patterned resist and the etching protection coat as masks, the substrate is etched with hydrofluoric acid solution, for example, to form a groove. Then, the resist and the protection coat are etched away. Also, in addition to the above described substrate, a substrate such as glass provided with through holes using a technique such as ultrasonic processing is prepared. Finally, after the substrate provided with grooves and the substrate provided with through holes are laminated with the groove being at the inner side thereof and the laminated substrates are heated, for example, in a vacuum furnace (in case both of them are glass plates, at around 600° C. for several hours), followed by leaving them to cool for fusion to produce the chip.
As described above, in case of glass, a groove must be formed on plane glass one by one to produce chips using a method as an extension of a technique for producing semiconductor integrated circuits (a combination of photolithography technique and etching technique). Also, in the process of production, many hazardous chemicals are used, and the production process takes long hours and requires expensive large equipment for use in production of semiconductors and the like. Furthermore, the above described chip made of glass has a disadvantage that it is apt to splinter and should be handled carefully.
Furthermore, for use in medical diagnosis and the like, the chip may be contacted by samples originated from patients such as blood, and it is desirable that the above described chip is made disposable, but glass material is incombustible, thus raising problems also in waste disposal. Therefore, it is not suitable for POC analyses etc. requiring inexpensiveness.
On the other hand, for medical diagnosis, the concentrations of a variety of substances in samples originated from biological bodies such as blood, urine and cerebrospinal fluid are widely detected quantitatively or qualitatively. Items to be detected in samples originated from biological bodies include the enzyme activity of GOT, GPT, γ-GTP and ALP, total cholesterol, triglyceride, glucose, hemoglobin Alc (HbAlc), and further proteins such as creatinine kinase, C reactive proteins (CRP) and cytokinins, antigens originated from bacteria and virus and antibodies against them.
Detection of these substances to be detected is performed by reacting the sample with an enzyme and antibody specific to the substance to be detected to thereby convert the substance ultimately to a substance (coloring dyes, fluorescent substances, luminescent substances, etc.) that can be detected by absorbance, fluorescence, chemical luminescence and so forth and determining the amount of the final substance (Ogawa, Z. et al., Clinical Investigation, 41:981 (1997), Kanno, T., Clinical Investigation, 42:309 (1998)).
These detection reactions are carried out by weighing a fixed amount of a sample and one kind or more of reagent solutions respectively and mixing them to implement reaction at a predetermined temperature for fixed time period.
In central laboratories of major hospitals and automatic analyzers adopted by clinical examination companies, a fixed volume or weight of samples and reagent solutions are weighed respectively with automatic pipettes. Also, in case of manual analysis, examiners weigh a fixed amount of samples and solutions using pipettes and quantitative capillaries.
Examination of contamination for food is performed in a similar way (Japanese Patent Laid-Open No. 4-64063, Method of Detecting Food Contaminating Bacteria).
In case of determining the amount of environmental pollutants, various kinds of reagents are often made to react using river water and soil extracts as samples to detect object substances (Japanese Patent Laid-Open No. 9-72898, Method of Analyzing Soils).
Methods in which these reactions for detection are carried out in the chip, that is, some reactive reagents and standard reagents are mixed with samples in the chip to implement reaction, and the post-reaction sample is analyzed include methods described below.
One of them is a method in which predetermined amounts of the sample and reagent solutions are weighed outside the chip and are then injected in the chip. Also, there is a method in which a predetermined volume of channel (reservoir) such as messcylinder is provided in the chip and delivered liquid is controlled accurately by means of combination of a pump with valves or applying electric field, thereby weighing and mixing the sample and reagent solution in the chip (for example, A. Manz et al., Trends Anal. Chem., Vol. 10, 144, 1991). Furthermore, there is a method in which the sample and reagent solution are poured into the chamber and are mixed to implement reaction, followed by weighing a fixed amount of them to separate components and analyzing quantitatively the amount of each of the separated components (S. C. Jacobson et al., Anal. Chem., Vol. 6, 4127,1994). In any of these methods, a process of weighing the sample and reagent solution or their mixture is required, and a method in which analysis is carried out while delivering liquid continuously at a constant flow rate ratio has not been proposed.
On the other hand, a concept of mixing two liquids in a predetermined ratio without weighing operations has also been proposed (U.S. Pat. No. 5,785,831 (HP), Japanese Patent Laid-Open No. 8-261986 (Japanese Patent of corresponding to U.S. Pat. No. 5,785,831)). However, the concept is to simply mix two liquids in the diverged channel, and it does not include a concept of carrying out predetermined chemical reaction continuously and using the reaction for detection of specific substances. Similarly, methods in which among two laminar flows contacting each other at a predetermined flow rate, interaction near the interface is used have also been proposed (WO 9739338, U.S. Pat. No. 5,716,852, WO 9747390). However, also in this case, it is basically means for extracting or measuring necessary molecules and particles using difference in diffusion rate due to difference in sizes of particles and molecules contained in each flow, and is not to implement predetermined chemical reaction.
Also, there are examples of carrying out necessary chemical reactions without weighing operations (J. Micromech, Microeng. 4, 246–256 (1994), Verpoorte E. M. J., //Manz A., deRooij N. F. INTERFACIAL DESIGN AND CHEMICAL SENSING, Chapter 21 pp. 244–254, America Chemical Society (1994)). That is, two or more chips made of silicon having grooves on the surface are overlaid on one another to form the capillary and reactive reagent solution is delivered by a pump to the capillary at a constant flow rate, thereby mixing the sample solution with the reactive reagent solution at a predetermined ratio and implementing reaction in the capillary.
However, in this method, the sample solution is simply mixed with the reactive reagent solution at a predetermined ratio, and for actual implementation processes, it is not substantially different from the batch system in which the sample solution and the reactive reagent solution are put in a mixing tank at a predetermined ratio.
Furthermore, in a structure like this having a plurality of chips overlaid on one another, a channel has a three-dimensional structure, thus making it difficult to go up step by step in the channel and obtain measured values at variety of reaction time. That is, quantification can be done at the endpoint of the enzyme reaction, but it is difficult to do quantification in a rate assay in which the amount of enzyme is determined from the reaction rate.
For analyzers, research and development aimed at POC analyses etc. is currently in progress, including the fact that chips comprising capillaries have been proposed. However, as described above, the material of chip comprising capillaries is generally glass and silicon to which fine processing can be applied with high accuracy. Therefore, the cost for processing is high and there are also disadvantages that the chip is apt to splinter and careful handling is required, and so on. Furthermore, for use in medical diagnosis and the like, the chip may be contacted with samples originated from patients such as blood, and it is thus desirable that the chip comprising a capillary is disposable, but glass material is incombustible thus raising problems also in waste disposal.
Also, considering an analyzer combining channel equipment and detection equipment, for a method using luminescent phenomena, light-intercepting efficiency is not high because emitted light is diverged in all directions.
Of methods using luminescent phenomena, chemical luminescent reactions have advantages of high speed and high sensitivity and of relatively inexpensive equipment because no light source is required for detection, but have disadvantages that luminescence is rapidly decayed, reagents are unstable, the background is high, and so forth.
Furthermore, in a similar way, the fluorescent method has an advantage that its reaction system has been known for a long time, but it requires as optical systems, excitation light sources and optical filters for separating excitation light from fluorescence, and so on.
Also, the fluorescent method is not suitable for the case in which a trace amount of sample in a fine capillary for use in the present invention is detected because the yield of emitting fluorescence is low and so on.
Also, the absorptiometric method has a disadvantage that it is necessary to make the length of optical path large so as to obtain accurate results and particularly long optical path is obtained for detecting a trace amount of samples, thereby making the structure of detection cells complicated, because the ratio of the incident light to the transmitted light is detected in principle.
In this way, for analyzers detecting a trace amount of sample in the fine capillary for use in the present invention, those that are easy for handling and economical, and are capable of performing analysis of high sensitivity, and can be downsized are not available, and analyzers suitable for POC analyses etc. are desired in the present circumstances.
On the other hand, detecting paper that enables the value of blood sugar and the like to be detected by dissolving solid reagents (freeze-dried reagents or paper and fiber impregnated with a predetermined amount of reagents) in the sample using only plasma are on the market. These solid reagents are convenient because it is not necessary to weigh reagents, but have a disadvantage that they are poor in quantitative accuracy compared to liquid reagents.
Furthermore, methods in which the sample and reagent are weighed outside the chip and are injected into the chip thereafter to implement reaction for detection not only require much manpower but also produce wastes in addition to wastes of chips. Also, in case man does not weigh the sample and reagent, a weighing system is required in addition to the chip, thus leading to large scale equipment as a whole. Furthermore, it is necessary to provide a channel in the chip for weighing the sample and reagent, thereby making the channel in the chip more complicated and leading to high costs. Also, these methods have a disadvantage that introduction of the operation of weighing the sample and reagent causes the process of analysis to be more complicated without distinction of inner and outer sides of the chip. Furthermore, the prior art requires additional means for adjusting timing for each process that is continuous and needs to control time accurately, because of the batch-type sample process and detection.